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Molecular indicators of Nitrobacter spp. population and growth activity during an induced inhibition event in a bench scale nitrification reactor

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Abstract

The Nitrobacter spp. ribosomal RNA gene (rDNA) and transcript (rRNAt) abundance were quantified in a bench scale nitrification reactor during baseline periods of high nitrification efficiency and an intervening staged inhibition event. The transcript to gene ratio (rRNAt/rDNA) was highly sensitive to changes in the reactor nitrite oxidation rate. During high nitrification efficiency, the rRNAt/rDNA metric displayed a range from 0.68 to 2.01 with one-sided (α=0.10) lower and upper prediction intervals of 0.70 and 1.78, respectively. When nitrification was inhibited by disabling the reactor pH control system, this activity metric declined an order of magnitude to ≈ 0.05, well below the lower prediction interval reflecting high nitrification efficiency. The decline was rapid (2h) and preceded a significant drop in reactor nitrification performance, which occurred as ammonia accumulated. The rRNAt/rDNA ratio remained low (≈ 0.05) for several days after the pH control system was re-enabled at a setpoint of 8.0, which otherwise induced rapid oxidation of accumulated ammonia and produced high free ammonia concentrations. The timing of a subsequent increase in the rRNAt/rDNA ratio, which transiently exceeded the upper prediction interval established during the baseline period of high nitrification efficiency, was not coincidental with resumption of pH control at 7.2 that lowered free ammonia concentrations to non-inhibitory levels. Rather, nitrite oxidation resumed and the rRNAt/rDNA ratio increased only after oxidation of accumulated ammonia was complete, which was coincidental with reduced reactor oxygen demand. In summary, the Nitrobacter rRNAt/rDNA activity metric reflected timely and easily recognizable changes in nitrite oxidation activity, illustrating that molecular data can be used to diagnose poor biological wastewater treatment performance.

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... However, in Isaka et al. (2007 studies, the immobilized AOB have grown at low temperature i.e. 5–10 °C (Isaka et al. 2007). Moreover, the autotrophic nitrification is commonly used in the wastewater treatment by autotrophic nitrifying bacteria like Nitrosomonas, Nitrococus and Nitrobacter which prefer the low temperatures of growing (4–10 °C) (Dinçer and Kargi 2000; Morozkina and Zvyagilskaya 2007; Zhang et al. 2011; Hawkins et al. 2012). Also, for a lot of denitrifying bacteria, the optimal temperature for their growth was ranged from 30 to 35 °C (Kim et al. 2006). ...
... Generally, for the Alcaligenes species, the optimum pH for their growth is between 6 and 7 (Wen and Wei 2011). Nevertheless, according to Hawkins et al. (2012), it was noted that for a fixed pH at 8.2, the nitrification effectiveness was reduced compared with pH 7 from 97 to 60 % (Hawkins et al. 2012 ). Moreover, Arthrobacter sp. ...
... Generally, for the Alcaligenes species, the optimum pH for their growth is between 6 and 7 (Wen and Wei 2011). Nevertheless, according to Hawkins et al. (2012), it was noted that for a fixed pH at 8.2, the nitrification effectiveness was reduced compared with pH 7 from 97 to 60 % (Hawkins et al. 2012 ). Moreover, Arthrobacter sp. ...
... However, in Isaka et al. (2007) studies, the immobilized AOB have grown at low temperature i.e. 5-10°C (Isaka et al. 2007). Moreover, the autotrophic nitrification is commonly used in the wastewater treatment by autotrophic nitrifying bacteria like Nitrosomonas, Nitrococus and Nitrobacter which prefer the low temperatures of growing (4-10°C) (Dinçer and Kargi 2000;Morozkina and Zvyagilskaya 2007;Zhang et al. 2011;Hawkins et al. 2012). Also, for a lot of denitrifying bacteria, the optimal temperature for their growth was ranged from 30 to 35°C (Kim et al. 2006). ...
... Generally, for the Alcaligenes species, the optimum pH for their growth is between 6 and 7 (Wen and Wei 2011). Nevertheless, according to Hawkins et al. (2012), it was noted that for a fixed pH at 8.2, the nitrification effectiveness was reduced compared with pH 7 from 97 to 60 % (Hawkins et al. 2012). Moreover, Arthrobacter sp. ...
... Generally, for the Alcaligenes species, the optimum pH for their growth is between 6 and 7 (Wen and Wei 2011). Nevertheless, according to Hawkins et al. (2012), it was noted that for a fixed pH at 8.2, the nitrification effectiveness was reduced compared with pH 7 from 97 to 60 % (Hawkins et al. 2012). Moreover, Arthrobacter sp. ...
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... However, in Isaka et al. (2007) studies, the immobilized AOB have grown at low temperature i.e. 5-10°C (Isaka et al. 2007). Moreover, the autotrophic nitrification is commonly used in the wastewater treatment by autotrophic nitrifying bacteria like Nitrosomonas, Nitrococus and Nitrobacter which prefer the low temperatures of growing (4-10°C) (Dinçer and Kargi 2000;Morozkina and Zvyagilskaya 2007;Zhang et al. 2011;Hawkins et al. 2012). Also, for a lot of denitrifying bacteria, the optimal temperature for their growth was ranged from 30 to 35°C (Kim et al. 2006). ...
... Generally, for the Alcaligenes species, the optimum pH for their growth is between 6 and 7 (Wen and Wei 2011). Nevertheless, according to Hawkins et al. (2012), it was noted that for a fixed pH at 8.2, the nitrification effectiveness was reduced compared with pH 7 from 97 to 60 % (Hawkins et al. 2012). Moreover, Arthrobacter sp. ...
... Generally, for the Alcaligenes species, the optimum pH for their growth is between 6 and 7 (Wen and Wei 2011). Nevertheless, according to Hawkins et al. (2012), it was noted that for a fixed pH at 8.2, the nitrification effectiveness was reduced compared with pH 7 from 97 to 60 % (Hawkins et al. 2012). Moreover, Arthrobacter sp. ...
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Agricultural activities lead excessive emission of ammonia nitrogen in the environment and can profoundly interfere the equilibrium of the natural ecosystems leading to their contamination. Actually, the biological purification of wastewaters is the most adopted technique thanks to its several advantages such as high performance and low energy consumption. For this reason, two novel strains of Alcaligenes sp. S84S3 and Proteus sp. S19 genus were isolated from an activated sludge and applied in the treatment of ammonium and nitrite in aqueous solution. Under the optimum operating conditions of temperature (30 °C), pH (7), carbon substrate (2 g/L of glucose) and duration of incubation time (69 h), the strain Alcaligenes sp. S84S3 could oxidize 65 % of the ammonium as high as 272.72 mg-NH4+/L. Moreover, during 48 h, the nitrate reduction rate performed by the strain Proteus S19 was about 99 % without production of nitrite intermediate (negligible concentration). Moreover, the coculture of the strains Alcaligenes sp. S84S3 and Proteus sp. S19 could eliminate 65.83 % of the ammonium ions without production of toxic forms of nitrogen oxides during a short time of incubation (118 h) at the same operational conditions with providing the aeration in the first treatment phase. The coculture of our isolated strains is assumed to have a good potential for nitrification and denitrification reactions applied in the treatment of wastewater containing ammonium, nitrite and nitrate. As a result, we can consider that the mixed culture is a practical method in the treatment of high-strength ammonium wastewater with reducing of sludge production.
... The relative abundance of 16S rRNA with respect to DNA, often presented as the 16S rRNA:16S rRNA gene ratio, is considered to be an indicator of microbial activity or potential of being active (Campbell et al., 2011;Perez-Osorio et al., 2010;Pitkanen et al., 2013;Kim and Wang, 2009;Campbell et al., 2009;Hawkins et al., 2012). To determine the rRNA abundance in a microbial sample, the 16S rRNA and 16S rRNA gene must be quantified separately. ...
... To determine the rRNA abundance in a microbial sample, the 16S rRNA and 16S rRNA gene must be quantified separately. A number of studies have applied both quantitative polymerase chain reaction (qPCR) and reverse transcription-qPCR (RT-qPCR) to examine the 16S rRNA:16S rRNA gene ratio for microbes from a variety of environmental systems, including plant leaves (Kim and Wang, 2009), marine surface waters (Campbell et al., 2011(Campbell et al., , 2009, biofilms (Perez-Osorio et al., 2010), nitrification reactor (Hawkins et al., 2012) and water from sewage treatment plants (Pitkanen et al., 2013). Most studies do not specifically report the extent of DNA or RNA losses during sample processing when determining the rRNA abundance, thus the accuracies of those reported ratios, which spanned almost 5 orders of magnitude from~0.1 (Hawkins et al., 2012) to~10 4 (Perez-Osorio et al., 2010), are largely unknown. ...
... A number of studies have applied both quantitative polymerase chain reaction (qPCR) and reverse transcription-qPCR (RT-qPCR) to examine the 16S rRNA:16S rRNA gene ratio for microbes from a variety of environmental systems, including plant leaves (Kim and Wang, 2009), marine surface waters (Campbell et al., 2011(Campbell et al., , 2009, biofilms (Perez-Osorio et al., 2010), nitrification reactor (Hawkins et al., 2012) and water from sewage treatment plants (Pitkanen et al., 2013). Most studies do not specifically report the extent of DNA or RNA losses during sample processing when determining the rRNA abundance, thus the accuracies of those reported ratios, which spanned almost 5 orders of magnitude from~0.1 (Hawkins et al., 2012) to~10 4 (Perez-Osorio et al., 2010), are largely unknown. In addition, high intra-sample variation, as indicated by large coefficients of variation (CV) within sample replicates, has also been observed in these studies (Perez-Osorio et al., 2010;Pitkanen et al., 2013;Kim and Wang, 2009;Campbell et al., 2009;Hawkins et al., 2012). ...
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... Though the influent pH led to an influent FA concentration of about 6-8 mg/L, which according to Anthonisen et al. (1976) could have led to NOO inhibition, NOO were active and nitrate production was observed from level L 1 in both STF reactors, e.g. of up to 12 ± 8 mg/L and 10 ± 1 mg/L for STF-1 and STF-2, respectively, during Phase II-D. These results are in accordance with the conclusion of Hawkins et al. (2012) that stated that FA does not significantly inhibits NOO; the NOO inhibition is mostly the result of poor competition for oxygen with a very active AOO population. ...
... However, the variation of ammonia oxidization rate at a constant NH 3 -N concentration and various pHs highlights the close dependence of nitrification process on pH. Hawkins et al. (2012) observed that when the pH decreased at values of 5.6, the nitrification activity was severely inhibited. Oxidation of ammonia is usually the ratelimiting step in the conversion of ammonia to nitrate. ...
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+ per litre has been developed at the Delft University of Technology. The SHARON process operates at a high temperature (30–40 °C) and pH (7–8). The process is performed without sludge retention. This enables the prevention of nitrite oxidation, leading to lower operational costs. Denitrification is used to control the pH. A full scale plant was designed (1500 m3) based on kinetic and stoichiometric parameters determined al 1.5.1. scale and model predictions. Total costs are estimated at about $1.7 per kg removed NH4+-N. The first full scale SHARON plant will be operational at the Dokhaven waste water treatment plant in Rotterdam in the beginning of 1998. This contribution focuses on the principles of the process and evaluates conditions for which application seems feasible.
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The Nitrobacter ribosomal RNA transcript (rRNAt) abundance reflected nitrite oxidizing activity in a bench-scale nitrification reactor (BSNR) biomass. A newly designed real-time PCR detection system targeting the Nitrobacter 16S-23S intergenic spacer region (ISR) was used to assess transcript levels. In a batch nitrite oxidation experiment, the Nitrobacter rRNAt abundance varied dramatically (32-fold) as the growth prospect changed from unfavorable (nitrite starvation: 4.21 +/- 0.85 X 10(8) copies/L), to favorable (excess nitrite: 1.35 +/- 0.24 X 10(10) copies/L), and back to unfavorable (nitrite starvation: 5.88 +/- 1.01 X 108 copies/L) over a 15-h time period. The Nitrobacter ribosomal gene (rDNA) abundance varied comparatively little (less than twofold) during this time. Further, when nitrifying activity was pH inhibited in the BSNR, the Nitrobacter rRNAt abundance varied significantly (35-fold) as the growth prospect changed from unfavorable (pH 6.2: 3.71 +/- 1.35 X 10(8) copies/L) to favorable (pH 7.2: 1.29 +/- 0.16 X 10(10) copies/L), while the Nitrobacter rDNA abundance again varied comparatively little (less than twofold). The results indicate that real-time PCR is better suited to measure activity rather than population changes in slow growing bacteria such as nitrite oxidizers. Utilization of rRNAt as an in situ measure of nitrite oxidizing activity could be used to improve the implementation of traditional nitrification, where high NOB activity is promoted, as well as alternate N-conversion pathways which suppress nitrite oxidation.
Article
We investigated the relationship between the growth rate and the ratio of RNA to DNA in of four resin acid degrading bacteria isolated from a sequencing batch reactor (SBR). Chemical assays as well as slot blot hybndizations with species-specific oligonucleotide probes were used to quantify the nucleic acids. These slow-growing bacteria have a positive linear correlation between growth rate and RNA/DNA ratio, similar to faster-growing bacteria like E. coli. We propose to use this correlation to measure metabolic activities of selected resin acid degrading bacteria in the complex community of the SBR in situ using species-specific hybridization probes. Preliminary experiments suggest that hybridization probes can be used to detect growth rate-dependent changes in the RNA/DNA ratio.
Article
Resin acids contribute to the acute toxicity of pulp and paper mill effluent. We monitored the abundance and metabolic activity of the resin acid degrader, Pseudomonas abietaniphila BKME-9, within complex communities of laboratory-scale effluent treatment systems using competitive PCR and competitive RT-PCR, targeting the 16S rDNA and rRNA. In continuous culture, a positive linear relationship was found between the rRNA:rDNA ratio and specific growth rate of BKME-9. In batch culture, the rRNA:rDNA ratio peaked briefly during early exponential growth of BKME-9. During continuous growth of an activated sludge community inoculated with BKME-9, the rRNA:rDNA ratio of BKME-9 decreased when the community was stressed with an alkaline pH shock. After the pH shock, the metabolic activity and population of BKME-9 recovered concomitantly with recovery of resin acid removal activity in the system. Thus, BKME-9 has characteristics that could be useful for bioaugmentation of biotreatment systems, and it is possible to monitor the population and activity of BKME-9 in such complex systems.
Article
The objective of the research program was to study process changes that could be used to maintain nitrite build-up and overcome the effects of acclimation to free ammonia (FA) and allow the use of a shortened nitrification/denitrification pathway for nitrogen removal from highly nitrogenous wastes. The study employed bench-scale, activated sludge cells. Measures investigated to overcome the effects of acclimation were: (1) increasing MLSS wastage by a factor of five; (2) extension of contact time to high FA levels to 50% of the total retention time; (3) raising FA concentrations to 40 mg NH3-N/l; (4) use of a more complex substrate (municipal landfill leachate); (5) double substrate inhibition (FA and sodium chlorate); (6) provision of internal denitrification; and (7) temporary reduction of FA levels. Of these, the most effective was internal denitrification.
Article
The anaerobic ammonium oxidation (Anammox) process, discovered 20 years ago, is, in combination with partial nitritation, ideally suited to treat nitrogen rich waste water streams such as digester effluent. In this review the engineering aspects and the practical application of the process are reviewed. The conventional nitrification–denitrification and nitritation–denitritation are also discussed briefly.The environmental conditions affecting the nitrification process, free ammonia and nitrous acid concentration, temperature, pH and dissolved oxygen concentration, are discussed. These conditions can be controlled in such a way that the partial nitritation step produces an Anammox-suited influent. The Anammox reactor conditions should favour the growth of the Anammox organisms in view of their low growth rate and possible inhibition effects. Dissolved oxygen and nitrite concentrations should be kept as low as possible and biomass washout should be limited. If the partial nitritation process and the Anammox process are occuring in the same reactor, care should be taken to the dissolved oxygen concentration, the ammonium load and the nitrite concentration to obtain a sustainable co-existence between aerobic and anaerobic ammonium oxidizers.An overview is presented of the practical implementation of autotrophic nitrogen removal. The process can be accomplished in the same reactor (1-reactor system) or by using 2 separate reactors (2-reactor system). Typically the 1-reactor system is a biofilm or granular reactor where the ammonium oxidizers are active in the outer layers of the biofilm or granule, producing a suitable amount of nitrite for the Anammox organisms that are active in the inner layers. Transport of ammonium and the produced nitrite is governed by diffusion. Finally, the different nitrogen removal processes are compared in terms of operational conditions and a direction for future work is provided.
Article
To understand how to optimize performance of a partially nitrifying plant, the dynamics of Nitrospira and Nitrobacter abundance were studied over a 1 year period using quantitative polymerase chain reaction (qPCR) and their relative contributions to nitrite oxidation assessed including the affects of temperature and dissolved oxygen (DO). Correlation coefficients linking shifts in the community composition of nitrite-oxidizing bacteria (NOB) to operational or environmental variables indicated Nitrospira was significantly and negatively correlated to nitrite concentrations (r = -0.45, P < 0.01) and DO (r = -0.46, P < 0.01), while temperature showed a strong positive correlation (r = 0.59, P < 0.0001). However, the Nitrobacter portion of the total NOB populations showed a positive correlations with DO (r = 0.38, P < 0.01) and hydraulic retention time (HRT) (r = 0.33, P < 0.05), as well as being negatively correlated with temperature (r = -0.49, P < 0.001) suggesting specific niche adaptations within the NOB community. Nitrospira was dominant being better adapted to the low DO and shorter sludge retention times (SRT) of this plant, while Nitrobacter increased in abundance during the winter months, when temperatures were lower and DO concentrations higher. Principal component analysis (PCA) results supported these findings by the close proximity of Nitrospira and temperature biplots of PC1 and PC2 as well as grouping Nitrobacter, NO(2)(-)-N, HRT, and DO in the loadings together. The clustering of samples from specific dates also exhibited a strong seasonality.
Article
Free ammonia has long been identified as a nitrite oxidation inhibitor. However, past attempts to use this compound to eliminate nitrite oxidation and thereby promote more efficient nitrogen removal strategies during biological wastewater treatment have often failed. Additionally, contradictory results exist in the literature where direct measurements of free ammonia inhibition of nitrite oxidation have been reported. In this study, suspended biomass samples (nitrifier enriched activated sludge) were collected from a bench scale nitrification reactor with Nitrobacter spp. as the dominant nitrite oxidizer and subjected to batch respirometric experiments designed to quantify free ammonia inhibition of nitrite oxidization. A variety of data including ammonia, nitrite, and nitrate conversion rates, oxygen consumption rates, and Nitrobacter ribosomal RNA transcript abundance, a molecular indicator of growth activity, were used to assess nitrite oxidation and growth activity. Both the traditional and molecular activity assessments indicated that free ammonia had a limited inhibitory effect on Nitrobacter spp. In fact, the pH changes necessary to induce high free ammonia concentrations (>10mg-N/L) had a demonstrably more important inhibiting effect on nitrite oxidation than free ammonia. In contrast, during high ammonia oxidizing activity (5.3mg-N/L/h), low nitrite oxidation rates (0.2mg-N/L/h) and severely impaired Nitrobacter spp. growth activity, indicated by a low abundance of the Nitrobacter spp. ribosomal gene transcript relative to the ribosomal gene (0.08), were measured. The findings suggest that pH changes and ammonia oxidizing bacteria activity are more important factors limiting Nitrobacter spp. mediated nitrite oxidation, rather than the free ammonia concentration.
Article
The goal of this work was to demonstrate the feasibility of treating leachate with high ammonium concentrations using the SBR technology, as a preparative step for the treatment in an anammox reactor. The cycle was based on a step-feed strategy, alternating anoxic and aerobic conditions. Results of the study verified the viability of this process, treating an influent with concentration up to 5000 mg N-NH(4)(+) L(-1). An effluent with about 1500-2000 mg N-NH(4)(+) L(-1) and 2000-3000 mg N-NO(2)(-) L(-1) was achieved, presenting a nitrite to ammonium molar ratio close to the 1.32 required by the anammox. Furthermore, taking advantage of the biodegradable organic matter, the operational strategy allowed denitrifying about 200 mg N-NO(2)(-) L(-1). The extreme operational conditions during the long-term resulted on the selection of a sole AOB phylotype, identified by molecular techniques as Nitrosomonas sp. IWT514.
Article
The research results clearly indicated the inhibitory effects of unionized ammonia (FA) and unionized nitrous acid (FNA) on the nitrification process. The FA and FNA concentrations, rather than total ammonia or nitrite ion concentrations, inhibit nitrification. The concentrations of FA that inhibit nitrosomonads are greater than those that inhibit nitrobacters. The ranges of FA concentrations that begin to inhibit the nitrifying organisms are: FA inhibition to nitrosomonads, 10 to 150 mg/l and FA inhibition to nitrobacters, 0.1 to 1.0 mg/l. The inhibition of nitrifying organisms was initiated at concentrations of FNA between 0.22 and 2.8 mg/l. The following may affect the inhibitory FA and FNA concentrations in a nitrification system; acclimation of the nitrifiers to FA and FNA, temperature, and the number of active nitrifying organisms. An operational chart may be used to assess the performance of nitrifying systems. Nitrification patterns may be modified by using operating procedures that increase or reduce the inhibitory effects of FA and FNA as appropriate. FA and FNA inhibition has been shown to occur with municipal, industrial, and agricultural wastes and with fertilizers in the soil.
Article
Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes to measure steady-state cellular pre-16S rRNA pools during growth state transitions in Escherichia coli. Pre-16S rRNA became undetectable when cells entered the stationary phase on rich medium and was replenished upon restoration of favorable growth conditions. These fluctuations were of much greater magnitude than concurrent fluctuations in the mature 16S rRNA pool. The extent of pre-16S rRNA depletion depended upon the circumstances limiting growth. It was significantly more pronounced in carbon-energy-starved cells than in nitrogen-starved cells or in cells treated with energy uncouplers. In the presence of the transcriptional inhibitor rifampin, rates of pre-16S rRNA depletion in carbon-energy-starved cells and nitrogen-starved cells were similar, suggesting that the difference between these conditions resides primarily at the level of pre-rRNA synthesis. Chloramphenicol, which inhibits the final steps in rRNA maturation, halted pre-16S rRNA depletion under all conditions. The data show that E. coli cells continue to process pre-rRNA after growth and rrn operon transcription cease, leading to drainage of the pre-rRNA pool. This supports the feasibility of using pre-rRNA-targeted probes to monitor bacterial growth in natural systems, with the caveat that patterns of pre-rRNA depletion vary with the conditions limiting growth.
Article
The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems in using cellular rRNA contents for direct monitoring of bacterial growth activity in situ. In a recent paper, Cangelosi and Brabant suggested monitoring the content of precursors in rRNA synthesis (pre-rRNAs) as an alternative approach. These are rapidly broken down after the cessation of bacterial growth. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coil cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coil growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA.
Article
The objective of this paper was to determine the best conditions for partial nitrification with nitrite accumulation of simulated industrial wastewater with high ammonia concentration, lowering the total oxygen needed in the nitrification step, which may mean great saving in aeration. Dissolved oxygen (DO) concentration and pH were selected as operational parameters to study the possibility of nitrite accumulation not affecting overall ammonia removal. A 2.5 L activated sludge reactor was operated in nitrification mode, feeding a synthetic wastewater simulating an industrial wastewater with high ammonia concentration. During the start-up a pH of 7.85 and a DO of 5.5 mg/L were used. The reactor was operated until stable operation was achieved at final nitrogen loading rate (NLR) of 3.3 kg N– NH4⁺/m³ d with an influent ammonia concentration of 610 mg N–NH4⁺/L.
Article
The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.
Article
Aerobic granules were successfully developed at substrate N/COD ratios ranging from 5/100 to 30/100 by weight. By measuring respective respirometric activities of heterotrophic, ammonia-oxidizing, and nitrite-oxidizing bacteria, it was found that the relative abundance of nitrifying bacteria over heterotrophs in aerobic granules was closely related to the substrate N/COD ratios. Results further showed that the populations of both ammonia and nitrite oxidizers were significantly enriched with the increase of the substrate N/COD ratio, while a decreasing trend of heterotrophic population was observed in the aerobic granules. These seem to indicate that high substrate N/COD ratio favors the selection of nitrifying bacteria in the aerobic granules, while the relative activity of nitrifying population against heterotrophic population evolved until a balance between two populations was reached in the aerobic granular sludge community. Moreover, cell elemental composition was correlated with the shift in microbial populations, e.g., the enriched nitrifying population in the aerobic granules resulted in a high cell nitrogen content normalized to cell carbon content. This study provides a good insight into microbial interaction in aerobic granules.
Article
Nitrogen can be eliminated effectively from sludge digester effluents by anaerobic ammonium oxidation (anammox), but 55-60% of the ammonium must first be oxidized to nitrite. Although a continuous flow stirred tank reactor (CSTR) with suspended biomass could be used, its hydraulic dilution rate is limited to 0.8-1 d(-1) (30 degrees C). Higher specific nitrite production rates can be achieved by sludge retention, as shown here for a moving-bed biofilm reactor (MBBR) with Kaldnes carriers on laboratory and pilot scales. The maximum nitrite production rate amounted to 2.7 gNO2-Nm(-2)d(-1) (3 gO2m(-3)d(-1), 30.5 degrees C), thus doubling the dilution rate compared to CSTR operation with suspended biomass for a supernatant with 700 gNH4-Nm(-3). Whenever the available alkalinity was fully consumed, an optimal amount of nitrite was produced. However, a significant amount of nitrate was produced after 11 months of operation, making the effluent unsuitable for anaerobic ammonium oxidation. Because the sludge retention time (SRT) is relatively long in biofilm systems, slow growth of nitrite oxidizers occurs. None of the selection criteria applied - a high ammonium loading rate, high free ammonia or low oxygen concentration - led to selective suppression of nitrite oxidation. A CSTR or SBR with suspended biomass is consequently recommended for full-scale operation.
Article
The Nitrobacter spp. rRNA gene (rDNA) and relative rRNA transcript abundance (rRNAt/rDNA ratio) were evaluated in response to sudden changes in the nitrite oxidation rate. The rDNA abundance poorly indicated sudden transitions in the rate, whereas the relative rRNAt abundance usually varied quickly and significantly. In response to changes in nitrite concentration, 8 h were required for the rRNAt/rDNA ratio to transition from a minimum value at nitrite starvation (approximately 0.07) to a maximum value with excess nitrite present (approximately 4), and 5 h were required for this metric to return to the minimum value after nitrite starvation re-ensued. Generally, the relative rRNAt abundance dropped significantly after 4.5 h of exposure to three different inhibitors. A sharp decline in the rRNAt/rDNA ratio occurred during exposure to 3,5-DCP (from 4 down to 0.2) even as the fractional inhibition level remained low (< 0.10); the minimum ratio value was observed when nitrite oxidation was completely inhibited. The ratio decreased significantly during exposure to azide (from 4 to 0.5) and H+ (from 2 to 0.2), but only when the fractional inhibition levels were high (> 0.8). Interestingly, when the pH was suddenly changed to 4.5, inhibiting nitrite oxidation completely, the rRNAt/rDNA metric did not decline suggesting that rRNAt processing was inhibited. This effect was not observed during severe inhibition with 3,5-DCP and azide. Overall, the findings indicate the relative rRNAt abundance can be used to closely track in situ Nitrobacter spp. activity and in most instances will reveal inhibition events with the potential to impact treatment performance in reactors where Nitrobacter spp. are dominant.
A targeted study on possible free ammonia inhibition of Nitrospira Elevated precursor 16S rRNA levels suggest the presence of growth inhibitors in wastewater
  • R A Simm
  • D S Mavinic
  • W D Ramey
Simm, R.A., Mavinic, D.S., Ramey, W.D., 2006. A targeted study on possible free ammonia inhibition of Nitrospira. Journal of Environmental Engineering and Science 5 (5), 365e376. Stroot, P.G., Oerther, D.B., 2003. Elevated precursor 16S rRNA levels suggest the presence of growth inhibitors in wastewater. Water Science and Technology 47 (11), 241e250.
  • C P L Grady
  • G T Daigger
  • H C Lim
Grady, C.P.L., Daigger, G.T., Lim, H.C., 1999. Biological Wastewater Treatment, second ed. Marcel Deffer, Inc., New York.